Tetanus toxin is internalized into neurons by way of endosomes and is not trafficked through the Golgi apparatus, demonstrated by studies with monensin, bafilomycin, and brefeldin A. The low pH of the endosome appears necessary for translocation of tetanus toxin to the neuronal cytosol. Immunoreactivity for VAMP/synaptobrevin, an integral membrane protein of synaptic vesicles thought to be the intracellular target of the catalytic domain of tetanus toxin, disappears from synaptic terminals in neuronal cultures treated with the toxin. Synaptic vesicles in toxin-blocked terminals dock but do not fuse with the presynaptic membrane. Five of the seven serotypes of botulinum toxin block the release of glycine and glutamate in spinal cord cell cultures. The rank order of potency in the culture assay is similar to that in mouse injection assays. Competitive binding studies indicate that the E, but not the B, serotype interferes with the binding of serotype A, suggesting some homology in the binding domain of serotypes A and E. A chimeric protein consisting of the transmembrane domain of anthrax toxin and the catalytic domain of tetanus toxin is cytotoxic (in the presence of the anthrax binding factor) to a number of non-neuronal cell lines, suggesting that the toxin acts to cleave other cellular proteins required for vesicle membrane fusion.